Abstract
This thesis contains two major topics. The first one is “Characterization of the histidine-containing phosphotransfer (Hpt) protein B□mediated multi-step phosphorelay system in Pseudomonas aeruginosa PAO1” and the second one is “The ATP-binding motif in AcoK is required for regulation of acetoin catabolism in Klebsiella pneumoniae CG43”. Pseudomonas aeruginosa is a gram-negative pathogen causing many acute and chronic infections, particularly in hospitalized individuals. It contains three genes that encode proteins with an Hpt domain but lack a kinase domain. Hpt proteins are signal mediators between hybrid sensors and response regulators. The proteins play a crucial role in directing signal transduction in bacteria, yeasts and plants. The study in first topic demonstrates that the Pseudomonas aeruginosa HptB-mediated signaling system consists of four orphan sensor kinases, HptB, and a specific response regulator, PA3346. We also present evidence of phosphatase activity of PA3346 on its neighboring gene product, PA3347. Finally, the swarming and biofilm forming activites of hptB, PA3346, and PA3347 knockout mutants are described. The second part of this thesis is to characterize a transcriptional factor AcoK in the regulation of acetoin catabolism. Many bacterial species utilize acetoin as a carbon source. The compound is oxidized by acetoin dehydrogenase encoded by acoABCD operon in Klebsiella pneumoniae. Previously, we have shown the expression of this operon is induced by acetoin through AcoK, the product of a gene located immediately upstream of acoABCD. AcoK contains a helix-turn-helix DNA binding domain of the LuxR transcription activator family at the C-terminal region and putative Walker A and B nucleotide binding motifs at N-terminal portion. The goal of the second study is to understand the contribution of different domains, in particular the nucleotide binding motif, in AcoK on its transcriptional activity. A number of truncations and site-directed mutations were constructed on AcoK and the biochemical and trans-activation activities of the resulting proteins were determined and reported herein. A mutation in the putative Walker A motif resulted in a significant reduction of ATP hydrolysis and trans-activation activity of AcoK on acoABCD expression, presumably was due to the loss of ATP-binding ability. The transcription factor bound specifically to a region comprising nucleotide -66 to -36 of the acoABCD promoter, although the DNA binding ability was not affected by the Walker A motif mutation. Together, this study provides an additional example in how a member of the Signal Transduction ATPases with Numerous Domains family activates its target gene expression.